Normal oxidative metabolism leads to the generation of various redox forms of molecular oxygen, termed reactive oxygen species (ROS), that are generated over a range of concentrations within cells. Low levels of ROS production are important for normal signaling mechanisms, while higher levels of ROS production can lead to oxidant stress, a condition under which the flux of ROS exceeds antioxidant capacity. A key determinant of the response to oxidant stress is the cell's capacity to counter increased ROS generation by adaptively increasing the production of NADPH, the principal source of reducing equivalents for the reduction of oxidized glutathione. The primary cytosolic enzyme required for NADPH synthesis is glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway. We have previously demonstrated the importance of this enzyme in maintaining normal endothelial function, including nitric oxide (NO) bioactivity, in the face of oxidant stress. In this proposal, we hypothesize that G6PD and its enzymatic product NADPH are the key regulators of the thiol redox state of the endothelial cell, are essential for normal oxidant signaling and endothelial function, adaptively respond to increased ROS generation to maintain a state of compensated oxidant stress, and when oxidant stress exceeds this adaptive capacity are responsible for the conversion of compensated oxidant stress to uncompensated oxidant stress in the endothelial cell. To test this hypothesis, we will 1) assess the relationship between NADPH and the redox state of endothelial thiol pools and their enzymatic determinants;2) evaluate the effect of NADPH on the synthesis, metabolism, and bioavailability of endothelial NO and its S-nitroso-derivatives;3) determine the role of G6PD and NADPH in the adaptive response to oxidant stress in endothelial cells, including their role in supporting the state of compensated oxidant stress;and 4) evaluate the relationship between thiol redox state and endothelial NO bioactivity under conditions of normal oxidant signaling, compensated oxidant stress, and uncompensated oxidant stress in vivo. These studies should shed light on the critical role of G6PD and NADPH in regulating the thiol redox state of the endothelial cell and its ability to adapt to oxidant stress in an effort to maintain normal endothelial function.